The type III secretion system (TTSS) is essential for the pathogenesis of a number of gram-negative human pathogens, including three Yersinia species;Y. pseudotuberculosis and Y. enterocolitica, that cause infection of intestinal lymphoid tissues, and Y. pestis, the etiological agent of bubonic and pneumonic plague. In Yersinia, the TTSS is composed of a needle-like structure, three translocator proteins involved in the delivery of toxins into the host cell (YopB, YopD and LcrV), and six effector proteins (YopE, YopT, YopH, YopJ, YopO, YopM) that target different host cell signaling molecules to disarm the innate and adaptive host immune system. YopB and YopD are thought to insert in the plasma membrane forming a translocation channel. Wild type Yersinia does not disrupt the integrity of the host cell membrane during the translocation process. Bacteria deficient in YopE and YopT, or expressing a form of the two proteins unable to down-regulate Rho GTPases, have strong pore forming activity. We have found that pore formation results from a Yersinia-induced host cell signaling that requires YopB and YopD, and that is blocked by inactivation of Rho GTPases. Here, we postulate that activation of such signaling pathways might also be required for an efficient translocation process. We will aim to determine a) what signaling events are required for pore formation, and whether those signals play a role in translocation;b) whether activation of additional host cell signals, such as the 21 integrin pathway, may be involved in pore formation or translocation;c) whether YopD may stimulate host-signaling functions by interacting with host cell partners. The type III secretion system (TTSS) is essential for the virulence of a number of gram- negative human pathogens of enormous clinical significance. Some of these organisms include important entropathogens such as Salmonella spp., Shigella spp., Yersinia enterocolitica, Y. pseudotuberculosis and enteropathogenic Escherichia coli as well agents causing severe pneumonia, like the opportunistic bacteria Pseudomonas aeruginosa, or the deadly Yersinia pestis. Interestingly, Y. pestis, the etiological agent of plague, and a currently considered a class A bioterrorism agent, is closely related to Y. pseudotubeculosis, which causes an infection limited to intestinal lymphoid tissues. The studies proposed in this application constitute the frame of an exploratory research project aim at testing a new hypothesis: that the TTSS of Yersinia manipulates the host cell machinery to inject its own toxins. This type III-mediated signaling may not be restricted to Yersinia and might take place during infection of host cells with other pathogens that encode homologs of proteins, such as enteropathogenic E. coli, Shigella, and Salmonella. Studies that answer these questions might shed light on the molecular nature of the complex interaction between bacterial pathogens bearing TTSSs and the host cell. Importantly, components that act by modulating the TTSS are potential targets for novel antimicrobials.